Adenovirus vectors have been extensively used as attractive vehicles to deliver foreign genes to a variety of cell types in vitro and in vivo.
Adenovirus lacks an envelope and is icosahedral in shape consisting of 252 capsomeres. Twelve capsomeres at each corner are called pentons (consisting of a penton base and fiber) having a protruding structure and the other 240 capsomeres are called hexons. Adenovirus penetrates into a cell (infection) by binding its fiber to a receptor CAR (for detail, see Bergelson J. M. et al., Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-1323, 1997), and by binding RGD motif of the penton base to integrin on the cell surface (Bai M, Harfe B, Freimuth P, Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells., J Virol 67: 5198-5205, 1993; Wickham T J et al., Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment. Cell 73: 309-319, 1993). When the virus reaches the endosome, the virus changes its conformation of the capsid protein under acidic conditions, thereby disrupting the endosome and penetrating into the cytoplasm. Since the first step is binding of the viral fiber to CAR which is a receptor on the cell surface, modifying the fiber may change the infection area of adenovirus vector (Paillard, F., Dressing up adenoviruses to modify their tropism. Hum Gene Ther 10:2575-2576, 1999).
A fiber gene is located in the L5 region of an adenovirus late gene. The fiber gene of adenovirus Type 5 consists of 581 amino acids and forms a trimer. The structure is composed of a tail, shaft, and knob part. A knob at the C-terminus binds to a receptor, CAR.
One of difficulties in the use of conventional adenovirus vectors is that the infection areas of the vectors have no tissue-specificity, and therefore the vectors transfer to many tissue cells non-specifically when they are administered systemically. Another difficulty is that the vectors cannot infect cells, which do not express adenovirus receptors (coxackievirus-adenovirus receptor (CAR); e.g., receptors for adenovirus Type 2 and 5 generally used as vectors for gene therapy. See Bergelson J. M. et al., supra for detail.).
Originally, adenovirus vectors, which have poly-lysine sequences having affinity for heparan sulfate at the C-terminus of the fiber molecule, have been constructed. These vectors have been reported to have wide infection area as expected (Wickham T J et al., Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins., J. Virol. 71:8221-8229, 1997; Yoshida Y et al., Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma., Hum Gene Ther 9:2503-2515, 1998; Gonzalez R et al., Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein., Gene Ther. 6: 314-320, 1999; Bouri K et al., Poly-lysine Modification of adenoviral fiber protein enhances muscle cell transduction., Hum Gene Ther 10: 1633-1640, 1999). However, it has been shown that insertion of a foreign peptide into the C-terminus of the fiber inhibits the fiber from forming a trimer; the vector has a viral titer 1 to 2 orders of magnitude less than that of a vector having wild type fibers; and the C-termrinus of the fiber is oriented towards the inside of the virus. Therefore, it is now thought that the region is not an optimal site for insertion of a foreign peptide.
In 1998, the group of Curiel et al. have directed their attention to the structure of the HI loop protruding from the viral surface, and have reported that insertion of a foreign peptide into the HI loop of the fiber enables exposure of the peptide on the viral surface, and this does not inhibit viral proliferation at all (Krasnykh V I et al., Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob., J Virol 72: 1844-1852, 1998; Dmitriev I et al., An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a Coxsackievirus and adenovirus receptor-independent cell entry mechanism., J Virol 72: 9706-9713, 1998). This report indicates a possibility that the HI loop of the fiber is an optimal expression site of a foreign peptide. However, they have constructed such a vector by the method using homologous recombination with special E.coli strain. Thus, the method is not simple enough to be widely used.